Non-intertwined strands of plasmid DNA contradicts the Watson and Crick model of DNA structure

According to Watson and Crick (W/C) model, a DNA molecule consists of two antiparallel polynucleotide chains, intertwined with each other. Although the W/C model is accepted widely, a number of researchers have raised questions against it and proposed alternative structures for DNA. In the present study, the W/C model was examined using plasmid DNA. It was hypothesized that two strands of plasmid DNA will remain intertwined and not separate from each other under denaturing conditions, if it follows the W/C model. To test this, plasmid DNA was denatured using sodium hydroxide (NaOH) and analyzed by gel electrophoresis. It was observed that addition of NaOH to pUC19 and pBR322 plasmids resulted in new form of DNA with higher electrophoretic mobility in agarose gel. DNA corresponding to higher electrophoretic mobility band of pUC19 (hmP19) was single-stranded and circular, indicating the separation of two strands of pUC19 plasmid. Next, we examined whether hmP19 DNA can re-anneal to form native pUC19 plasmid. It was observed that neutralization of NaOH resulted in the appearance of native pUC19 plasmid in denatured DNA. Native pUC19 was also formed by hmP19 DNA extracted from agarose gel and was found to be digestible with Hind III. Ability to confer ampicillin resistance in transformed demonstrated the Escherichia coli functionality of pUC19 plasmid formed by extracted hmP19 DNA. Reversible separation of two strands of plasmid into single-stranded circular DNA shows that DNA strands are not intertwined with each other and contradicts the W/C model of DNA structure.


Introduction
DNA is the genetic material of all organisms, with the exception of some viruses. The currently accepted model of DNA structure was proposed by James Watson and Francis Crick in 1953 1 . According to this model, a DNA molecule consists of two antiparallel polynucleotide chains, intertwined with each other. Although the Watson and Crick (W/C) model is accepted widely, some researchers have raised questions against it and proposed alternative models for DNA structure 2,3 . Among these, Rodley's model which envisaged that two strands of a DNA molecule are held side-by-side has generated significant interest and curiosity in the scientific community 2 .
In the present study, the W/C model of DNA structure was examined with the help of plasmid DNA. It was hypothesized that two strands of a plasmid will remain intertwined and not separate into single-stranded circular DNA molecules under denaturing conditions, if it follows the W/C model. To test this, pUC19 and pBR322 plasmids were denatured using sodium hydroxide (NaOH) and analyzed by gel electrophoresis. Interestingly, addition of NaOH to pUC19 and pBR322 plasmids resulted in new form of DNA showing higher electrophoretic mobility in agarose gel. DNA corresponding to higher electrophoretic mobility band of pUC19 (hmP19) was found to be singlestranded and circular, suggesting the separation of two strands of plasmid DNA. Under suitable conditions, hmP19 DNA re-annealed to form native pUC19 plasmid. These results showed that two strands of a DNA molecule are not intertwined with each other and contradicted the W/C model of DNA structure.

Methods
Plasmid isolation, denaturation and agarose gel electrophoresis pUC19 and pBR322 plasmids were isolated from E. coli strains [cultured in Luria Bertani broth (HiMedia, catalogue# M1245) in a shaking incubator at 37°C] by alkaline-lysis method as described previously 4 . For denaturation, approximately 5 µg plasmid DNA (concentration determined using NanoDrop spectrophotometer) was added with an equal volume (5 µl) of NaOH solution of indicated concentration.
DNA extraction from agarose gel hmP19 DNA bands were cut with the help of a clean knife. DNA was purified using an extraction kit, as suggested by manufacturer (FairBiotech, catalogue# DE0100). Briefly, gel was dissolved in DE buffer and passed through a column. After washing with buffers, DNA was eluted in 50 µl nuclease free water.

Bacterial transformation
Transformation of E. coli strain DH5-alpha with gel-extracted plasmid DNA was carried out by heat-shock method (42°C for 30 sec) using water bath. Transformed and non-transformed bacteria were spread on ampicillin-nutrient agar plates supplemented with 25 µl X-Gal (Himedia, catalogue# MB0690. Plates were kept overnight in a 37°C incubator.

Results and discussion
Two strands of a DNA molecule are held together by non-covalent interactions, which can be disrupted by increasing the pH of DNA solution 5 . In the present study, approximately 5 µg of plasmid DNA was denatured by adding NaOH solution of increasing concentration. It was observed that addition of 0.5 N NaOH to pUC19 resulted in a new form of DNA showing higher electrophoretic mobility in agarose gel (Figure 1a, Underlying data 6 ). Similar results were obtained with pBR322 plasmid added with 0.5 N NaOH (Figure 1b, Underlying data 7 ). Formation of higher electrophoretic mobility DNA in pBR322 plasmid added with NaOH has also been reported previously 8 .
DNA corresponding to higher electrophoretic mobility band of pUC19 (hmP19) was characterized using DNA modifying enzymes in next experiments. Incubation with Hind III, which acts on double-stranded DNA, digested pUC19 plasmid but not hmP19 DNA (Figure 1c, Underlying data 9 ). S1 nuclease, which digests single-stranded DNA, degraded hmP19 DNA but not pUC19 plasmid (Figure 1d, Underlying data 10 ). Exonuclease I and alkaline phosphatase, which would digest single-stranded linear DNA, degraded neither of pUC19 or hmP19 DNA (Figure 1e, Underlying data 11 ). These results showed that hmP19 DNA is single-stranded and circular. Exonuclease I-and alkaline phosphatase-digested primers (experimental control) did not form product in PCR reaction (Supplementary Figure 1, Extended data 12 ).
We asked whether hmP19 DNA was generated by separation of two strands of pUC19 or breakage of one strand followed by release of another one. Formation of a single band of higher electrophoretic mobility by denatured pUC19 (instead of two, which would have been the case when one strand was linearized and another was circular) suggested that hmP19 DNA was generated by separation of two strands of the plasmid (Figure 1a, b). To confirm this notion, hmP19 DNA was subjected to renaturing conditions. If hmP19 DNA were formed due to separation of two strands of pUC19, it would reanneal to form Figure 2. Single-stranded circular hmP19 DNA annealed to form double-stranded pUC19 plasmid. pH of denatured pUC19 plasmid DNA was normalized using HCl solution of indicated concentration. Agarose gel electrophoresis showed the formation of native pUC19 plasmid DNA in denatured plasmid DNA solution added with 0.25 M HCl (a). hmP19 DNA was extracted from agarose gel and approximately 3 µg of it was rerun on 1% agarose gel. Band pattern of pUC19 plasmid formed by extracted hmP19 DNA was same as that of native pUC19 plasmid (b). Gel-extracted hmP19 DNA was incubated with Hind III and run on 1% agarose gel. Hind III digested the pUC19 plasmid formed by gel-extracted hmP19 DNA (c). E. coli strain DH5-alpha was transformed with gel-extracted hmP19 DNA by heat-shock method. hmP19 DNA-transformed (d), but not non-transformed (e) bacteria formed colonies on ampicillin-X-Gal-nutrient agar plates. Representative data of two-three independent experiments are shown. the native plasmid under suitable conditions. Interestingly, neutralization of NaOH with 0.25 M HCl resulted in the appearance of pUC19 plasmid in denatured DNA solution (Figure 2a, Underlying data 13 ). To further confirm this, hmP19 DNA was extracted from gel and re-examined by gel electrophoresis. Both, gel-extracted hmP19 DNA and native plasmid DNA exhibited similar band patterns in the agarose gel ( Figure 2b). These results showed that single-stranded circular hmP19 DNA was formed Figure 1. Double-stranded plasmid is denatured by NaOH into single-stranded circular DNA. Approximately 5 µg plasmid DNA was added with an equal volume (5 µl) of NaOH solution of indicated concentration (normality, N) and run on 1% agarose gel. A distinct band of higher electrophoretic mobility DNA (marked by arrows) was observed in pUC19 (a) and pBR322 (b) plasmids added with 0.5 N NaOH. For enzymatic digestion, NaOH in denatured plasmid was neutralized using 0.5M HCl, followed by incubation with different enzymes. Native pUC19, but not hmP19 DNA was digested by Hind III, which acts on double-stranded DNA (c). S1 nuclease, which recognizes singlestranded DNA, degraded hmP19 DNA but not native pUC19 plasmid (d). Exonuclease I (Exo I) and alkaline phosphatase (AP), which eliminate single-stranded linear DNA, digested neither of native pUC19 or hmP19 DNA (e). Representative data of at least three independent experiments are shown.
due to reversible separation of two strands of pUC19 plasmid DNA.
Next, we characterized pUC19 plasmid formed by reannealing of gel-extracted hmP19 DNA. Interestingly, similar to the native plasmid, pUC19 plasmid formed by gel-extracted hmP19 DNA was also degraded by Hind III (Figure 2c). Functionality of pUC19 formed by gel-extracted hmP19 DNA was demonstrated by its ability to transform E. coli. hmP19 DNA-transformed bacteria acquired ampicillin resistance and formed colonies on ampicillin-nutrient agar plates supplemented with X-Gal (Figure 2d, Underlying data 14 ). No colonies were formed by non-transformed bacteria (Figure 2e). These results showed that pUC19 plasmid formed by re-annealing of hmP19 DNA was structurally and functionally similar to native plasmid DNA.
Concludingly, reversible separation of two strands of plasmid DNA into single-stranded circular DNA molecules shows that DNA strands are not intertwined with each other. These findings contradict the W/C model of DNA structure and provide evidence for the side-by-side structure of DNA.

Grant information
The author(s) declared that no grants were involved in supporting this work.
Plasmid DNA molecules have been determined by researchers to be circular, double stranded helical structures. In this work, however, the author claims plasmid DNA molecules follow a nonhelical model, supported by experimental results that showed alkali-denatured plasmid DNA is not intertwined but separated. The author further showed separated single stranded plasmid DNA renatured to form native plasmid DNA at neutral pH that could transform E. coli bacteria.
This article challenges the helical DNA model of plasmid DNA accepted by the mainstream scientific community. However, the claim that plasmid DNA is not intertwined cannot be supported by the experimental results presented and the logic in the article is flawed.
The agarose gel electrophoresis was the only method used to show separation of plasmid DNA strands. Figures in the article showed that alkali-denature plasmid DNA resulted in a band of higher mobility, which could only be digested by S1 nuclease that degraded single stranded DNA. However, the appearance of a higher-mobility band could not prove that the two single strands of were separated and not intertwined. It was totally possible that the denatured DNA strands did not form helices but still had a part intertwined to each other to keep them from separating. Such intertwined DNA could also be digested by S1 nuclease but not Hind III or exonuclease. Furthermore, since the two strands might not be separated, they could easily re-hybridize to form native plasmid DNA in the neutralized pH. Thus, the author's claim that the two strands of the plasmid were separated was untenable.
The helical structure of plasmid DNA has been supported by many studies, some of which also used denatured plasmid DNA and presented results directed contradicted to the claims of this article. JiaYu et al. showed alkali-denatured plasmid could either remain supercoiled doublestranded or became single-stranded but not separated with atomic force microscopy, both of which showed increased mobility in agarose gel. Lifeng Yan and Hiroshi Iwasaki 1 showed similar structures with AFM after thermal denaturation. The phenomenon that the two strands of plasmid DNA could not be separated under denaturing conditions has been demonstrated with electron microscopy and other techniques decades ago 2 . 34 The author should comment on those papers in this article when proposing a contradicting model.

1.
Enzymatic digestions performed in this study are done in the presence of NaOH in a number of cases, where the pH of the solution would be ~13 going by the concentrations given by the author. At this pH will the enzymes HindIII, S1 nuclease and Exo1+AP function at all? Since, the bands observed in these gels in the presence of NaOH is used as an observation to support the hypothesis of the study. This is a concern that must be addressed by the author.

2.
In Fig 1d, the author claims that S1 nuclease digests single stranded DNA in the right most lane (7th from left, 1st lane ladder) while double-stranded remains intact. Going by this argument, why hasn't the enzyme chewed up the NaOH denatured DNA (hmP19) band in the 6th lane?

3.
Additional point is that, the gel isn't capable of providing the information of what is the nature of the hmP19 band. It could very well be that the tertiary structure of the puC19 is altered in the presence of NaOH with Watson-Crick pairs remaining to a significant extent forming something like a super-supercoiled state of DNA. The assumption that hmP19 is a single stranded state seems a little far fetched with only gel based assays.

4.
One thing that I am unable to wrap around is that, if the two single strands are indeed not paired by Watson The author is trying to argue the Watson-Crick Model with his experimental result observed from the faster mobility of denatured plasmids. Unfortunately, his explanation is completely wrong and cannot support his conclusion for four main reasons: The higher electrophoretic mobility band of plasmids (hmpUC19 or hmpBR322) is actually random coils of the NaOH denatured plasmids composed of a pair of single stranded circular DNAs (ssc DNA). In alkaline solution, the denatured plasmids are paired ssc DNAs; they are probably tightly tangled with each other just without hydrogen bonds. In 0.25 N NaOH, the backbone of the plasmid is stable and keeps intact, however, due to topological rule, the paired ssc DNAs cannot separate from each other; they just tangled with each other. As soon as the paired ssc DNAs entered into agarose during agarose gel electrophoresis (AGE), the sudden pH change pushes the denatured ssc DNAs renature quickly but not in legitimate way i.e., adopting many inter-strand or intra-strand hydrogen bonds between the AT or GC pairs, causing the formation of tightly tangled entity with electric mobility higher than their supercoiled counterparts. The differences of the denatured plasmid in solution and in agarose gel are not commonly noticed by many scientists. It is reasonable and unquestionable that the NaOH denatured plasmids can renatured under suitable conditions as the author has indicated. It does not mean the alkaline denatured complementary ssc DNAs has been completely physically separated. 1.
If the phenomenon of higher mobility plasmid is really separated ssc DNAs, as the author supposed to be, they should be separable by AGE. The figure 3  ) indicated that the mobility of denatured plasmid is closely related to the supercoiling of plasmid, the alkaline denature relaxed plasmids moves much faster than that of their highly supercoiled counterparts. All native plasmids are composed of a set of covalently closed circular DNA (ccc DNA), the author cannot provide a reasonable explanation on how the paired ssc DNAs overcome the topological rule without the help of strand passing ability of topoisomerases.

4.
What the DNA really is? Many different answers can be heard from different observers. Just as a cubic or cylinder shaped iceberg seen by observers cannot jump to the conclusion that bottom of the iceberg is in the same shape.
The DNA structure has been studied by many scientists all over the world for many decades. Although many facts and experimental results contradicted or collided with one of the claims of the Once the double helix conjecture was proven, it would be a shock to the molecular biology because it confirmed the prediction of the ambidextrous model is correct and its meaning can be easily understood by anybody with normal IQ. Therefore, it may induce a "Paradigm shift" and maintain lasting influence on the tertiary structure of DNA. This meme will be transferred generations after generations since knowledge cannot inherit from parents; every student has to learn from the simplest beginning. As textbooks are the main source of their knowledge, it would be guilty if we keep on teaching them the Watson-Crick Model that we know is wrong or questionable.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com Page 14 of 14